Molecular Anatomy of a Trafficking Organelle

Microglia Promote Learning-Dependent Synapse Formation through Brain-Derived Neurotrophic Factor

This review is focused on purinergic neurotransmission, i.e., ATP released from nerves as a transmitter or cotransmitter to act as an extracellular signaling molecule on both pre- and postjunctional membranes at neuroeffector junctions and synapses, as well as acting as a trophic factor during development and regeneration. Emphasis is placed on the physiology and pathophysiology of ATP, but extracellular roles of its breakdown product, adenosine, are also considered because of their intimate interactions. The early history of the involvement of ATP in autonomic and skeletal neuromuscular transmission and in activities in the central nervous system and ganglia is reviewed. Brief background information is given about the identification of receptor subtypes for purines and pyrimidines and about ATP storage, release, and ectoenzymatic breakdown. Evidence that ATP is a cotransmitter in most, if not all, peripheral and central neurons is presented, as well as full accounts of neurotransmission and neuromodulation in autonomic and sensory ganglia and in the brain and spinal cord. There is coverage of neuron-glia interactions and of purinergic neuroeffector transmission to nonmuscular cells. To establish the primitive and widespread nature of purinergic neurotransmission, both the ontogeny and phylogeny of purinergic signaling are considered. Finally, the pathophysiology of purinergic neurotransmission in both peripheral and central nervous systems is reviewed, and speculations are made about future developments.

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Physiology and Pathophysiology of Purinergic Neurotransmission

We used an antiserum against purified cholinergic synaptic vesicles from Torpedo and expression screening to isolate a cDNA clone encoding synuclein, a 143 amino acid neuron-specific protein. A cDNA clone was also isolated from a rat brain cDNA library that encodes a highly homologous 140 amino acid protein. The amino terminal 100 amino acids of both proteins are comprised of an 11 amino acid repeating unit that contains a conserved core of 6 residues. The synuclein gene is expressed only in nervous system tissue, not in electric organ, muscle, liver, spleen, heart, or kidney. In the electric organ synapse Torpedo synuclein-immunoreactive proteins are found in 3 major molecular-weight classes of 17.5, 18.5, and 20.0 kDa. In the neuronal cell soma the 17.5 kDa species is predominant and immunoreactivity is localized to a portion of the nuclear envelope.

https://www.jneurosci.org/content/jneuro/8/8/2804.full.pdf

Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal

We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.

/pdf/synaptic-vesicle-exocytosis-captured-by-quick-freezing-and-1qgnb5cggj.pdf

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.

The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation

When brain tissue is homogenized in media iso-osmotic to plasma, the club-like presynaptic nerve endings resist disruption and are snapped or torn off from their attachments to form discrete particles (nerve-ending particles) in which all the main structural features of the nerve ending are preserved. For these particles we propose the name ' synaptosomes' in order to emphasize their relative homogeneity and their resemblance in physical properties to other subcellular organelles. They can be separated as a distinct fraction by differential and density-gradient centrifuging (Gray & Whit-Since acetylcholine is now well established, by all the classical criteria, as a central as well as a peripheral transmitter (for a review see Gaddum, 1961), it seems reasonable to conclude that the particle-bound acetylcholine and choline acetyltransferase of the fraction represent acetylcholine and enzyme localized within synaptosomes derived from chol-inergic neurones. Similarly, the 5-hydroxytrypt-amine and noradrenaline in this fraction are probably due to the presence of synaptosomes derived from neurones containing these amines. This view has been strengthened by the findings of Carlsson, Falck & Hillarp (1962), who have obtained histo-chemical evidence for the neuronal localization of these amines in the brain. The nerve endings and the synaptosomes derived from them have a complex fine structure when examined under the electron microscope with positive staining and thin sectioning (see review by Whittaker & Gray, 1962) or negative staining (Home & Whittaker, 1962). They are seen to consist (Plate 1 d) of thin-walled bags containing cytoplasm packed with synaptic granules or; frequently (though not in this example) one or more mitochondria are also present. The region of the post-synaptic membrane immediately adjacent to the ending is thickened; on homogenization it may remain adherent and accompany the synaptosome through the various steps of the fractionation procedure. Synaptic vesicles appear to be of at least three main kinds: 'hollow', 'dense-cored' and 'compound' (Plate 1 d). They have been proposed as the actual binding sites of transmitters within the nerve endings (De and as the morphological counterpart of the 'quantized' release of acetylcholine detected electrophysiologically (Fatt & Katz, 1952). The dense-cored vesicles are numerous in peripheral adrenergic nerve endings, and have there been proposed as the binding sites of noradrenaline. We have for some time been studying the disruption of synaptosomes with the object of obtaining , as separate fractions, synaptic vesicles, intraneuronal mitochondria, external and post-synaptic membranes and the soluble constituents of the nerve-ending cytoplasm for …

The separation of synaptic vesicles from nerve-ending particles (`synaptosomes')

1. Synaptic vesicles separated by density-gradient centrifugation from extracts of the cholinergic nerve terminals of the electric organ of Torpedo marmorata were found to contain appreciable amounts of ATP as well as acetylcholine. 2. Vesicular ATP was stable in the presence of concentrations of apyrase and myokinase that rapidly destroyed equivalent amounts of endogenous or added free ATP; pre-treatment of cytoplasmic extracts of electric tissue with these enzymes destroyed endogenous free ATP, but did not affect the vesicular ATP. 3. When [U-14C]ATP was added to electric tissue at the time of comminution and extraction of the vesicles, all the radioactivity was associated with soluble components in the subsequent fractionation: none was associated with vesicles or membrane fragments; thus it is unlikely that vesicular ATP can be accounted for by the sequestration of endogenous free ATP within any vesicles formed during comminution and extraction of the tissue. 4. When synaptic vesicles were passed through iso-osmotic columns of Bio-Gel A-5m, which separates vesicles from soluble proteins and small molecules, all the recovered ATP and acetylcholine passed through together in the void volume. 5. Regression analysis showed that vesicular ATP content was highly correlated with vesicular acetylcholine content in different experiments, the molar ratio acetylcholine/ATP being 5.32±(s.e.m.) 0.45 (21 expts.) for the peak density-gradient fraction. The ratio varied, however, somewhat across the density-gradient peak suggesting some degree of chemical heterogeneity in the vesicle population.

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles.

WHEN brain tissue is homogenized under carefully controlled conditions, a high proportion of the presynaptic nerve terminals are torn away from their attachments to form discrete particles for which the term ‘synaptosomes’ has recently been adopted (WHITTAKER, MICHAELSON and KIRKLAND, 1964). These particles may be separated from other subcellular brain particles by differential and density gradient centrifuging. They retain all the structural features of the nerve endings, together with their content of transmitter substances. On suspension in hypotonic media, a proportion of them swell and burst, discharging the contents of their cytoplasm. A simple density gradient procedure enables the various components of the synaptosome-soluble cytoplasmic constituents, synaptic vesicles, external membranes and intra-terminal mitochondriato be separated from the disrupted synaptosomes in relatively pure form, thus enabling the compartmentation of transmitter substances and other constituents of the nerve ending to be determined (WHITTAKER et al., 1963, 1964). Synaptosomes were first prepared by HEBB and WHITTAKER (1958) and first identified as such by GRAY and WHITTAKER (1960, 1962; see also WHITTAKER, 1960). The effect of hypotonic disruption was studied by WHITTAKER (1959, 1961), JOHNSTON and WHITTAKER (1963), DE ROBERTIS, RODRIGUEZ DE LORES ARNAIZ, SALGANICOFF, PELLEGRINO DE IRALDI and ZIEHER (1963) and WHITTAKER et al. (1964) (definitive papers only are quoted). The subject has been comprehensively reviewed by WHITTAKER (1964 a, b). This paper describes some further observations which have been made on the morphology and acetylcholine content of pure preparations of isolated synaptic vesicles. In a study of the properties of synaptosomes isolated from various areas of the central nervous system, regional differences in stability were observed during exposure to hypotonic conditions. Synaptosomes from the cerebral cortex were found to be more readily disrupted than those from some other areas and to give a higher yield of synaptic vesicles. Accordingly, cortical tissue was used as the source of synaptic vesicles in the present study. Methods have been devised for determining the average number of acetylcholine molecules/vesicle and the findings are discussed in relation to the problem of the quantized release of acetylcholine at cholinergic nerve endings. Preliminary accounts of this work have been given by SHERIDAN and WHITTAKER (1964) and WHITTAKER (1 964 c).

Victor P. Whittaker

Papers

The isolation of nerve endings from brain: An electron microscopic study of cell fragments derived by homogenization and centrifugation

The separation of synaptic vesicles from nerve-ending particles (`synaptosomes')

Adenosine triphosphate. A constituent of cholinergic synaptic vesicles.

The morphology and acetylcholine content of isolated cerebral cortical synaptic vesicles.

The preparation and characterization of synaptic vesicles of high purity.